Kinases and protein kinase cascades are involved in most cell signaling pathways, and many of these pathways play a role in human disease. For instance, kinases have been implicated in cell entry into apoptosis [P. Anderson, Micobiol. Mol. Biol. Rev., 61, pp. 33-46 (1997)], cancer [P. Dirks, Neurosurgery, 40, pp. 1000-13, (1997)], Alzheimer's disease [K. Imahori et al., J. Biochem., 121, pp. 179-88 (1997)] angiotensin II and hematopoietic cytokine receptor signal transduction [B. Berk et al., Circ. Res., 80:5, pp. 607-16 (1997); R. Mufson, FASEB J., 11:1 pp. 37-44 (1997)], oncoprotein signaling and mitosis [A. Laird et al., Cell Signal, 9:3-4 pp. 249-55 (1997)], inflammation and infection [J. Han et al., Nature, 386 296-9 (1997).] An understanding of the structure, function, and inhibition of kinase activity could lead to useful human therapeutics.
The structures of a number of protein kinases have been solved by X-ray diffraction and analyzed [reviewed in L. Johnson et al., Cell, 85, pp. 149-158 (1996); E. Goldsmith et al., Cur. Opin. Struct. Biol., 4, pp. 833-840 (1994); S. Taylor et al., Structure, 2, pp. 345-355 (1994)]. Enzymes in the kinase family are often characterized by two domains separated by a deep channel. The N-terminal domain creates a binding pocket for the adenine ring of ATP, and the C-terminal domain contains the presumed catalytic base, magnesium binding sites, and phosphorylation lip. Sequence homology among the kinases varies, but is usually highest in the ATP-binding site. ATP is a substrate common for all kinases.
Among medically important tyrosine kinases are epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR), Flk-1, and src.
One particularly important class of serine/threonine kinases are the mammalian mitogen-activated protein (MAP)1 kinases. These kinases mediate intracellular signal transduction pathways [M. H. Cobb et al., J. Biol. Chem., 270, pp. 14843-6 (1995); R. J. Davis, Mol. Reprod. Dev., 42, pp. 459-67 (1995)]. Members of the MAP kinase family share-sequence similarity and conserved structural domains, and include the extracellular-signal regulated kinases (ERKs), Jun N-terminal kinases (JNKs) and p38 kinases. JNK and p38 kinases are activated in response to the pro-inflammatory cytokines TNF-α and interleukin-1, and by cellular stress such as heat shock, hyperosmolarity, ultraviolet radiation, lipopolysaccharides and inhibitors of protein synthesis [B. Derijard et al., Cell, 76, pp. 1025-37 (1994); J. Han et al., Science, 265, pp. 808-11 (1994); J. Raingeaud et al., J. Biol. Chem., 270, pp. 7420-6 (1995); L. Shapiro et al., Proc. Natl. Acad. Sci. U.S.A., 92, pp. 12230-4 (1995)]. In contrast, ERK kinases are activated by mitogens and growth factors [D. Bokemeyer et al., Kidney Int., 49, pp. 1187-98 (1996)].
ERK2 is found in many different cell types. ERK2 is a protein kinase that achieves maximum activity when both Thr183 and Tyr185 are phosphorylated by the upstream MAP kinase kinase, MEK1 [N. G. Anderson et al., Nature, 343, pp. 651-3 (1990); C. M. Crews et al., Science, 258, pp. 478-80 (1992)]. Upon activation, ERK2 phosphorylates many regulatory proteins, including the protein kinases Rsk90[C. Bjorbaek et al., J. Biol. Chem. 270, pp. 18848-52 (1995)] and MAPKAP2 [J. Rouse et al., Cell, 78, pp. 1027-37 (1994)], and transcription factors such as ATF2 [J. Raingeaud et al., Mol. Cell. Biol., 16, pp. 1247-55 (1996)], Elk-1 [J. Raingeaud et al. (1996)], c-Fos [R. H. Chen et al., Proc. Natl. Acad. Sci. U.S.A., 90, pp. 10952-6 (1993)], and c-Myc [B. L. Oliver et al., Proc. Soc. Exp. Biol. Med., 210, pp. 162-70 (1995)]. ERK2 is also a downstream target of the Ras/Raf dependent pathways [S. A. Moodie et al., Science, 260, pp. 1658-61 (1993)] and may help relay the signals from these potentially oncogenic proteins. ERK2 has been shown to play a role in the negative growth control of breast cancer cells [R. S. Frey et al., Cancer Res., 57, pp. 628-33 (1997)] and hyperexpression of ERK2 in human breast cancer has been reported [V. S. Sivaraman et al., J. Clin. Invest., 99, pp. 1478-83 (1997)]. Activated ERK2 has also been implicated in the proliferation of endothelin-stimulated airway smooth muscle cells, suggesting a role for this kinase in asthma [A. Whelchel et al., Am. J. Respir. Cell. Mol. Biol., 16, pp. 589-96 (1997)]. In addition, ERK2 appears to be involved in platelet-derived growth factor-directed migration of vascular smooth muscle cells, suggesting that this kinase may be also be involved in restenosis and hypertension. [K. Graf et al., Hypertension, 29:1, pp. 334-339 (1997)].
The crystal structures of unphosphorylated p38 [K. P. Wilson et al., J. Biol. Chem., 271, pp. 27696-700 (1996); Z. Wang et al., Proc. Natl. Acad. Sci. U.S.A., 94, pp. 2327-32 (1997); (Brookhaven PDB entry, 1WFC)], and ERK2 [F. Zhang et al., Nature, 367, pp. 704-11 (1994); (Brookhaven PDB entry, 1ERK)] have been solved.
Recently, a phosphorylated ERK2 crystal structure has also been solved [B. J. Canagarajah et al., Cell, 90, pp. 859-69 (1997)]. The fold and topology of ERK2 is similar to p38 [K. P. Wilson et al. (1996)], and the two proteins are 48% identical in amino acid sequence.
p38 was identified as a kinase that was phosphorylated on tyrosine following stimulation of monocytes by LPS [J. C. Lee et al., Nature, 372, pp. 739-46 (1994)]. p38 kinase was cloned [J. Han et al. (1994)] and shown to be the target for pyridinylimidazole compounds that block the production of IL-1β and TNF-αby monocytes stimulated with LPS [J. C. Lee et al. (1994)]. SB203580, a 2,4,5-triarylimidazole, is a potent p38 kinase inhibitor that is selective relative to other kinases, including other closely related MAP kinases [A. Cuenda et al., FEBS Lett., 364, pp. 229-33 (1995); A. Cuenda et al., EMBO J., 16, pp. 295-305 (1997)]. The structure of SB203580 in complex with p38 has been reported [L. Tong et al., Nat. Struct. Biol., 4, pp. 311-6 (1997)]. The crystal structure of a different pyridinylimidazole compound, VK-19,911,4-(4-fluorophenyl)-1-(4-piperidinyl)-5-(4-pyridyl)-imidazole in complex with p38 has also been described [K. P. Wilson et al., Chem.& Biol., 4, pp. 223-231 (1997)]. These structures identified the residues important for binding pyridinyl-imidazoles, and revealed that both compounds bind within the ATP binding site of p38. Many of these residues are conserved in ERK2, but there are enough differences that binding of pyridinyl-imidazole compounds does not occur. A similar situation exists for JNK3, which also shares structural similarity to p38, but is unable to bind pyridinyl-imidazole inhibitors. This same to type of scenario, wherein a compound binds to one family member, but not to the majority of others, is also likely to occur in other serine/threonine kinase and tyrosine kinase families.
However, the kinase family members that do not share affinity for a compound that binds to one member may be equally, if not more important from a medical standpoint. Thus, there is an ongoing need to identify potential inhibitors of those other kinases.